The mechanical-double enzyme method was used in the current study to isolate and culture primary chondrocytes from the chicken growth plates. The feasibility and practicability of the approach were determined by using trypan blue staining, toluidine blue staining, PCR, and flow cytometry. The immunofluorescence assay was also used to effectively identify chondrocytes, demonstrating the expression of chondrocyte-specific secreted products (Col-II and Aggrecan). The exterior morphology of chondrocytes was studied at several stages, revealing significant changes in cell shape with each generation. Notably, compared to earlier approaches, the mechanical-double enzyme strategy revealed enhanced cell adhesion and much reduced apoptosis rates. The findings indicate that this novel method has great potential for efficient primary chondrocytes culture, providing important insight into chondrocyte ba research and future applications in cartilage tissue engineering. The following technical points are included in this method:•Isolation and culturing primary chondrocytes by a mechanical-double enzyme approach.•The evaluation of cell adhesion and apoptosis of mechanical double enzyme approach as compared to previous approaches.•The confirmation of chondrocyte-specific secreted products' expression via toluidine blue staining, PCR, and immunofluorescence assays.
Keywords: Cartilage; Chondrocyte; Mechanical Double Enzyme Technique for chondrocytes; Mechanical-double enzyme.
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